Development and evaluation of a real-time EBOV-L-RT-qPCR for detection of Zaire ebolavirus

Jääskeläinen, Anne J.; Moilanen, Kirsi; Aaltonen, Kirsi; Putkuri, Niina; Sironen, Tarja; Kallio‐Kokko, Hannimari; Vapalahti, Olli

doi:10.1016/j.jcv.2015.04.003

Cited by 16 publications

(10 citation statements)

References 4 publications

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“…In all the assays, two vials of each standard were reconstituted using 1 mL of molecular grade water, and viral RNA was extracted using commercially available kits. In-house assays were developed using published primers and probe sequences for the Ebola virus np gene [ 10 ] and l gene [ 18 ]. Ten-fold serial dilutions of the high concentration standards showed good efficiencies and linearity of the qRT-PCR for both targets ( Fig 2A and 2B ).…”

Section: Resultsmentioning

confidence: 99%

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

et al. 2015

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The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

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Section: Resultsmentioning

confidence: 99%

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

et al. 2015

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“…There are a growing number of molecular tests described in the literature. The tests that were selected in this study have been widely used not only in epidemic situations but also in external quality assessment studies [9][10][11][12][13][14][15][16][17][18][19][20]. Dual-target assays are increasingly used in commercial and in-house assays [6,[21][22][23].…”

Section: Discussionmentioning

confidence: 99%

Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome

et al. 2019

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Preparedness and response actions to mitigate Ebola virus disease (EVD) outbreaks rely on rapid diagnosis to be implemented locally to sort suspect patients attending health centers. Our aim was (i) to develop and evaluate an RT-qPCR assay combining primers and probes derived from two reference assays targeting different genomic regions; (ii) to study whether sensitivity and specificity of this dual-target assay were at least equal or better to the parental assays; (iii) to implement this dual-target assay onto the Cepheid GeneXpert open cartridge as a proof of principle for technological transfer aiming at bedsite testing locally. To do so, three home-made published RT-qPCR assays were selected to be compared with the RealStar® Filovirus Screen RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany), a technique that was largely deployed during the 2014–2015 West African EVD outbreak. Primers and probes sequences of the custom-made assays were analyzed in silico against a multiple sequence alignment, including >250 complete sequences corresponding to strains that have caused EVD epidemics in the past. Genomic RNA purified from the Mekambo strain of Zaire ebolavirus (EBOV) was used to study the sensitivity of the five methods. Based on these results, two in-house methods were selected and adapted to design the dual-target assay, which performances were compared to those of the parental assays using a synthetic RNA control. The dual-target assay showed better sensitivity and limit of detection (LoD95 at 0.4 copies/µL) than the parental methods (1.7 and 2.2 copies/µL). Ultimately, the dual-target assay was transferred onto the GeneXpert Flex-03 open cartridge, demonstrating a LoD95 at 0.75 copies/µL. Together these results indicate that EBOV dual-target assay has the potential to be used during EVD outbreak in the laboratory having performed molecular testing during the recent outbreaks.

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“…Total RNA was extracted using Direct-Zol RNA columns (Zymo Research Corp), and cDNA transcribed using Superscript III (Invitrogen). Samples were screened for filoviruses using four assays: 1) a nested filovirus ‘family level’ consensus PCR (cPCR) targeting a 680 bp fragment of the filovirus L gene [ 9 ], 2) an Ebolavirus ‘genus level’ cPCR targeting a 187 bp fragment of the NP gene [ 19 ], 3) a real-time PCR specific for the EBOV virus, targeting the L-gene [ 20 ], and 4) a real-time PCR specific for the BOMV virus, targeting the L-gene [ 9 ]. Samples were also screened using broadly reactive cPCR assays for corona [ 21 , 22 ], paramyxo [ 23 ], flavi [ 24 ], and influenza [ 25 ] viruses.…”

Section: Methodsmentioning

confidence: 99%

Spillover of ebolaviruses into people in eastern Democratic Republic of Congo prior to the 2018 Ebola virus disease outbreak

Goldstein

Belaganahalli

Syaluha³

et al. 2020

One Health Outlook

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Background The second largest Ebola virus disease (EVD) outbreak began in the Democratic Republic of Congo in July 2018 in North Kivu Province. Data suggest the outbreak is not epidemiologically linked to the 2018 outbreak in Equateur Province, and that independent introduction of Ebola virus (EBOV) into humans occurred. We tested for antibodies to ebolaviruses in febrile patients seeking care in North Kivu Province prior to the EVD outbreak. Methods Patients were enrolled between May 2017 and April 2018, before the declared start of the outbreak in eastern DRC. Questionnaires were administered to collect demographic and behavioural information to identify risk factors for exposure. Biological samples were evaluated for ebolavirus nucleic acid, and for antibodies to ebolaviruses. Prevalence of exposure was calculated, and demographic factors evaluated for associations with ebolavirus serostatus. Results Samples were collected and tested from 272 people seeking care in the Rutshuru Health Zone in North Kivu Province. All patients were negative for filoviruses by PCR. Intial screening by indirect ELISA found that 30 people were reactive to EBOV-rGP. Results were supported by detection of ebolavirus reactive linear peptides using the Serochip platform. Differential screening of all reactive serum samples against the rGP of all six ebolaviruses and Marburg virus (MARV) showed that 29 people exhibited the strongest reactivity to EBOV and one to Bombali virus (BOMV), and western blotting confirmed results. Titers ranged from 1:100 to 1:12,800. Although both sexes and all ages tested positive for antibodies, women were significantly more likely to be positive and the majority of positives were in February 2018. Conclusions We provide the first documented evidence of exposure to Ebola virus in people in eastern DRC. We detected antibodies to EBOV in 10% of febrile patients seeking healthcare prior to the declaration of the 2018–2020 outbreak, suggesting early cases may have been missed or exposure ocurred without associated illness. We also report the first known detection of antibodies to BOMV, previously detected in bats in West and East Africa, and show that human exposure to BOMV has occurred. Our data suggest human exposure to ebolaviruses may be more frequent and geographically widespread.

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Development and evaluation of a real-time EBOV-L-RT-qPCR for detection of Zaire ebolavirus

Cited by 16 publications

References 4 publications

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

Development and Evaluation of a Duo Zaire ebolavirus Real-Time RT-PCR Assay Targeting Two Regions within the Genome

Spillover of ebolaviruses into people in eastern Democratic Republic of Congo prior to the 2018 Ebola virus disease outbreak

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